During each division, the cell must quickly and accurately replicate its genome. This process, however, is challenged by constant insults to DNA. DNA interstrand cross-links (ICLs) are particularly toxic genomic lesions that covalently link the two strands of DNA. If left unrepaired, these lesions can block replication and induce genomic instability, a hallmark of cancer. Although ICLs are generated by a variety of exogenous and endogenous agents, the structures of specific ICLs that arise spontaneously in cells are unknown. In proliferating cells, ICL repair occurs predominately in S phase. In the classic ICL repair pathway, repair requires replication fork convergence at an ICL and the cross-linked DNA strands are unhooked by nucleolytic incisions that generate a DNA double stranded break (DSB) intermediate. This DSB is then repaired by homologous recombination. Importantly, mutations in genes that function in this repair pathway cause the bone marrow failure and cancer predisposition syndrome Fanconi anemia (FA). Recently, we discovered an alternative ICL repair pathway that depends on the NEIL3 DNA glycosylase. Like the FA pathway, the NEIL3 pathway is activated by ubiquitylation of the replicative CMG helicase upon fork convergence at an ICL. However, unlike the FA pathway, the NEIL3 pathway does not involve formation of a DSB intermediate. Instead, NEIL3 unhooks ICLs by cleaving one of the N-glycosyl bonds of the crosslinked nucleobases, generating an abasic site that can be bypassed by translesion synthesis. Unhooking by the NEIL3 pathway is therefore faster and less complicated than unhooking by the FA pathway and is the preferred ICL repair pathway for a subset of lesions. In this proposal, complementary biochemical and analytical approaches will be used to investigate the mechanism of NEIL3-dependent ICL repair. Aim 1 seeks to determine how replication forks activate NEIL3- dependent unhooking using Xenopus egg extracts that recapitulate ICL repair. Aim 2 proposes to investigate the dynamics of NEIL3 at individual replication forks using single molecule approaches. Finally, Aim 3 will address the question of which endogenous forms of DNA damage are targeted by ICL repair pathways through the development of a novel mass spectrometry approach to discover DNA lesions in cells. By understanding the mechanisms of ICL repair, it may be possible to design interventions that sensitize cancer cells to chemotherapy or mitigate the molecular defects that cause FA and other diseases.